Our primary aim is to examine the regulation of the secondary normal humoral immune response in humans. Our prior studies showed that individuals booster immunized with tetanus toxoid generate multiple subsets of antitetanus toxoid antibody-producing cells, each with presumably unique functions. Furthermore, a portion of the IgG-Tet-producing cells are highly sensitive to in vitro inhibition of function by treatment with native antigen. A similar refractive state can be induced in vivo by repetitive immunization. These studies indicate that B-cell hyporesponsiveness is a natural event of normal secondary immune responses and may indicate a locus for acquired or induced abnormalities. This proposal will further explore these inhibitory phenomena and in particular will determine: (1)\valency of the signals needed to be delivered to the B-cell for inducing tolerance; (2)\the role of suppressor T cells in the maintenance of the tolerant state; (3)\the molecular mechanisms for the establishment and maintenance of the tolerant state; and (4)\the relation of in vitro-generated B-cell tolerance to in vivo hyporesponsiveness. Initially, we will extend our previous finding that tetanus toxoid inhibition of IgG-Tet production requires only 1 hour of antigen contact with the B cells, whereas anti-isotype antibody inhibition requires at least 24 hours. The prediction is that the difference in time required for inhibition relates to the valency of the stimulus, i.e., univalent for antigen causing rapid tolerance and divalent for antibody causing less-rapid tolerance. We will test this by using conditions which make the antigen divalent and the antibody monovalent. In the second portion, we will further explore our observation that the tolerant state is not irreversible but in fact that the responsive state can be regenerated by removal of suppressor T cells. Studies in this project will examine the appearance in the circulation, the specificity and the maturity of these cells. In the third part of this project, we will primarily use metabolic inhibitors and stimulants to better elucidate how, on a molecular level, the tolerant state is generated, maintained and relaxed. Last, in the past several years we have shown that a hyporesponsive state can be also generated in vivo by multiple immunizations. In the latter portions of this project we will examine the role of B-cell tolerance in the maintenance of this hyporesponsive state.